Bone marrow is the preferred source for isolation of rat neutrophils and the subsequent acquisition of neutrophil extracellular traps

BACKGROUND: Neutrophil extracellular traps (NETs) are a network structure with DNA as skeleton scaffolding a wide range of antimicrobial proteins, which have been shown to contribute to the pathogenesis, persistence, and progression of many chronic inflammatory diseases. The method for isolation of human or mouse NETs has been well-established. However, in some diseases such as atrial fibrillation, the model can only be established in rats. While most related studies isolate rat neutrophils from the peripheral blood, which is insufficient for further acquisition of NETs. Despite the consumptiveness of rat peripheral blood neutrophil isolation, bone-marrow deprived neutrophil is rarely employed and has not been compared to peripheral neutrophil with its NETs secreting capability. METHODS: In the current study, a different bone-marrow-oriented strategy was described and conducted. Based on centrifugal program settings, a one-step method and a two-step method was developed and compared. The purity of the isolated neutrophils was determined by Wright’s staining and the viability was detected by flow cytometry. NETosis is the specialized cell death of neutrophils accompanied with NETs formation and its degree of rat neutrophils was analyzed by phase contrast microscopy, fluorescence microscopy, and Celigo whole view analysis. The Picogreen dsDNA Assay Kit was used to determine the concentration of NETs obtained from the NET-rich supernatants. The levels of secreted NETs in rat peripheral blood and bone marrow were compared. RESULTS: Approximately 0.5×10(8)–1×10(8) neutrophils could be obtained from the bone marrow of a single rat, with viability above 90%, which was comparable to that of neutrophils isolated from humans and mice. The final concentration of NETs obtained from the supernatant ranged from 8–12 µg/mL. The secretion of NETs from bone marrow-derived neutrophils showed a similar trend to polymorphonuclear (PMN) leukocytes. In addition, the extrusion of the intracellular matrix was incomplete during NETosis, and rat NETs showed weak cross-linking capability for forming large-scale net-like structures. CONCLUSIONS: The bone marrow-oriented strategy for isolating rat neutrophils is accessible and repeatable. NETs formation capability is similar between rat bone-marrow and peripheral blood neutrophils.